Haemostasis-3 Dr. Léránt István

Sept. 14, 2015.

Department of Medical Biochemistry Semmelweis University

Haemostasis III

ISTVÁN LÉRÁNT lerant.istvan@med.semmelweis-univ.hu

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3

Fibrinolysis

• Dissolution of fibrin network

– Plasmin

• Fibrinpolymer FDP (Fibrin degradation produts)

• Dissolution of thrombus.

4

FG sol

FN gel

D

E

D

FDP

Water soluble

Fibrinolysis

5

FG Water soluble

FN Water insoluble

D

E

D

FDP

Water insoluble

Ext. Int.

VIIa - TF XII

Blood coagulation

Plasminogen activators

Fibrinolysis

Fibrinolysis

Fibrinolysis

6

Plasminogen

Plasmin

Fibrin Fibrin degradation

products

t-PA

urokinase

PAI-1 -2

Thrombin TAFI

Factor XIIa, XIa,

Kallikrein

a2-antiplasmin

a2-macroglobulin

Streptokinase

7

8

Plasmin – digestion of fibrin

Plasmin

D E D

D E

D E

Monomer

F

FxxF D Dimer

9

Formation of blood clots

Thrombin

Factor XIII Faktor XIIIa

Inh.

Inh.

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Formation of blood clots

Thrombin

Factor XIII Faktor XIIIa

Inh.

Inh.

FDP

Time

F

FxxF

FxxFxxInh

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Formation of blood clots

Thrombin

Factor XIII Factor XIIIa

Inh.

Inh.

FDP

Time

F

FxxF

FxxFxxInh

Plasmin

Inh.

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PLASMINOGEN

Plasmin/ogen

• Liver

• Plasminogen - Plasmin

• t1/2 2,2 days

• [Plasminogen] 0,2 g/l

• Serinprotease

• Degradation of fibrin

• Kollagenase

• Mediators of complement

system

• ovulation

• fibronection

• Thombospondin

• Laminine

• Von-Willendbrand factor

• Monocytes

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Plasmin/ogen

• Activation

– t-PA

– Urokinase - uPA

– Streptokinase

– Fibrin

– Factor XII

• Inaktivation

– .a2-antiplasmin

– . a2-makroglobulin

– Aprotinin

– . eACA

– p-Aminomethylbensoic acid

• [plasmin]

– Thrombosis

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15

Glu-PLAsMINOGeN

K1

K2

K3

K4

K5

Lys77 Glu1

Arg561

S

S

Plasmin

Plasminogen Activator

Glu-PLASMIN

Lys-PLASMINOGEN

MINI-PLASMINOGEN

des-KRINGLE 1-4

ELASTASE

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Activation of plasminogen

Pln

PA

Glu-Plg

Proteolysis

Lys-Plg

Pln

PA

Conformational change

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Activation of plasminogen

Pln

PA

Glu-Plg

Proteolysis

Lys-Plg

Pln

PA

Conformational change

Time

Plasmin

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PLASMIN

• Primer function: – Fibrinonlysis

• t-PA – tissue Plasminogen Activator

• u-PA – urokinase

– Inhibitors • plasminogen activator inhibitor

(PAI)-1

• plasminogéen activator inhibitor PAI-2.

– The cell receptors of u-PA • Transmembran proteins,

• Signal transduction – nucleus

• Gene expression

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PLASMIN

• Activation of plasmin – Cell movement

– repair

– metastasis

• t-PA – Cognitive memory

– neurodegeneration

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Tissue-type plasminogen activator, t-PA

• Intrinzic activator of fibrinolysis

• serinprotease

• 527 amino acids

• Thrombolysis – Alteplase

– (rt-PA)

– Recombinant variants

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t-PA

Synthesis

Liver

Tumorcells

Active

cofactors

FDP

Myosin

Aktin

EC Membran

Polylysine

Denatured proteins

22

Single-chain t-PA

Synthesis

EC

Tumorcells K

S S

K AC

K

S

S AC

K Two-chain t-PA

t-PA

Plasmin

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Activation kinetics

Time

Plasmin activation

t-PA + FDP

t-PA

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Alteplase

• Myocardial infaction

– Recanalisation -

• Acute pulmonary embolism

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Urokinase-Typ Plasminogen Aktivator (uPA)

• Peptidase activity • Treatment – myocardial infarction

• Pulmonary embolims

• Urine - isolation

• Plasminogen – plasmin – conversion in circulation

• Inhibition of urokinase by PAI-1, PAI-2

• Cancer – Level of urokinase and PAI-1 is significantly elevated

– Marker - prognosis

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Single-chain urokinase

scu-PA

Synthesis

EC

Tumorcells

K

K

S S

Pn

K

S

S

Two-chain urokinase

tcu-PA

UROKINASE

AC

Lys158-Ile159

Fibrinolysis – feed back reactions

Urokinase

(scuPA)

Endothelcells

Tumorcells

Urokinase

(tcuPA)

Plasminogen

Plasmin

Plazmin

+ feed back

Endothelcells

Tumorcells

tPA

(sctPA)

t-PA

(tctPA)

Plazmin

?

Ineffective

+ Fibrin, Miosine, Actin, denatured proteins

Effective activation of plasminogen Endothelcells

Surface

tPA+ Plg

Fibrinolysis

• Initiation:

– t-PA – Plasminogen-Plasmin

• Amplification

– Prourokinase – uPA – Plasminogen-Plasmin

• PLASMIN

• Dissolution of Fibrinogen

– t-PA: cofactor – is dissolved

• Plasmin + inhibitors

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Streptokinase

• Bacterila proteins

• Antibody formation

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Streptokinase

Plasminogen Plasmin

Streptokinase

Fibrinogen Fibrin

SK

SK-PLG COMPLEX *

Plg Pln

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Inhibitors of fibrinolysis activators

• Inhibitors

• Plasminogenactivator inhibitors – PAI-1

– PAI-2

– PAI-3

– PAI-4

• PAI-1 – „main” inhibtoros

• Inhibition of t-PA and urokinase

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Inhibitors of fibrinolysis activators

• Activation of trombocytes

• PAI-1 release

• Thrombus - increase in level of PAI-1

• Enhancement of stability of thrombus

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PLAsMINOGeN-AcTIVaTOR-INHIBITOR-1 (PAI-1)

• SERPIN family

– MM.: 52.000 Da

– glycoprotein

– 379 Amino acids

• Inhibition of t-PA-t & tcu-

PA-t

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PLAsMINOGeN-AcTIVaTOR-INHIBITOR-1 (PAI-1)

• Secretion – hormons

– Cytokines

• 3 forms • Aktive

• Inaktive

• Latent

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PLAsMINOGeN-AcTIVaTOR-INHIBITOR-1 (PAI-1)

• SZINTesis

• Endothel cells

• megakariocytes

• adipocytes

• Thrombocytes • 80 % of the blood PAI-1

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PLAsMINOGeN-AcTIVaTOR-INHIBITOR-1 (PAI-1)

• [PAI-1]

– In obesity

– DM

– Atherosklerosis

• Risk of thrombus formation

• Embolism

• Infarktion

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a2-PLASMIN INHIBITOR

• Liver

– MM.:70000 Glycoprotein

– 13 % carbohydrates

– 452 Aminoacids.

• Plasmaconcentration 1M

• Inactivation of Plasmin

• k= 400*104M-1sec-1

• Inactivation of Plasmin

• (Fibrin)

– k= 1*104M-1sec-1 Plazmin

a2-antiplasmin

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a2-PLAZMIN INHIBITOR

• Complex of Plasmin- a2-

PLAsMIN INHIBITOR

• Inhibiton of adsorption

of plasminogen on

fibrin

• In Fibrinnetwork:

FxxFxxaAP

Plasmin

a2-antiplasmin

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a2-PLAZMIN INHIBITOR

• a2-Plazmin Inhibitor

– C-terminale Lys452

– reversible

– Plasmin LBS-1 Domain

• Inhibitors

– Lysin

– eACA

• Ser-OH

• Acyl-enzyme intermedier

Plasmin

a2 - Antiplasmin

452

Lys

SerOH

Arg354-Met355

eACA

Fibrinolysis & aPC + TM

Trombin

Trombin+TM Trombinként

inaktiív

Trombin+TM

Protein C/Ca

Faktor Va

Faktor VIIIa

TAFI Inhibition of

clot dissolution

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Fibrinolysis & aPC + TM

FIBRINOGEN

FAKTOR XIII

PLATELETS

COFACTOR V

COFACTOR VIII

FACTOR XI

THROMBINF

PROTEIN C

ENDOTHELSEJTEK THROMBOMODULIN

THROMBINB

COFAKTOR Va

COFAKTOR VIIIa

a

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TAFI

Thrombin activated fibrinolysis

inhibitor

Fibrinolysis & aPC + TM

a2 – Macroglobulin

• 3 M

• liver

• 4 subunits

• -S-S- bridges

• Protease –

• „Bait” regio

• Protease+a2M

• makrofag receptors

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Antitrombin/heparin ( 4 M)

• Fibrinolysis Faktoren

• Fibrinolysis

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t-PA uPA

Plazminogén

Plazmin

Fibrin

FDP

D-dimer

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Endothel-

cells

PAI-1

t-PA +PAI-1

Hepatocytes

Pln - PI

PI